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The effects of naltrexone on the development of physical dependence on morphine
European Journal of Pharmacology, 50 (1978) 193--202
193
© Elsevier/North-Holland Biomedical Press
THE EFFECTS OF NALTREXONE ON THE DEVELOPMENT OF PHYSICAL DEPENDENCE ON MORPHINE HEMENDRA N. BHARGAVA
Department of Pharmacognosy and Pharmacology, College of Pharmacy, University of Illinois at the Medical Center, Chicago, Illinois 60612, U.S.A. Received 20 January 1978, revised MS received 13 April 1978, accepted 20 April 1978
H.N. BHARGAVA, The effects of naltrexone on the development of physical dependence on morphine, European J. Pharmacol. 50 (1978) 193--202. A single i.p. injection of naltrexone (20 mg/kg) partially inhibited the development of physical dependence upon morphine in mice rendered dependent on morphine by implantation of a pellet containing 75 mg of morphine free base for three days. This was evidenced by an increase in the dose of naloxone (EDs0) required to precipitate withdrawal jumping response. The increase in naloxone EDs0 was much more pronounced when naitrexone was given prior to and during the course of pellet implantation. Inhibition was also observed when naltrexone was administered one day after the morphine pellet implantation, i.e., after some dependence had already developed. Naltrexone administration prior to and during the development of dependence also inhibited, but only partially, the loss in body weight and hypothermic response observed during abrupt withdrawal of morphine in morphine-dependent mice. The inhibitory effect of naltrexone on morphine dependence development was not associated with changes in brain morphine concentration. Brain morphine concentration Morphine dependence
Body weight Hypothermia
1. Introduction The mechanisms involved in the genesis of tolerance and physical dependence on narcotics are still obscure. Narcotic antagonists have been used as pharmacological tools to understand these phenomena. Orahovats et al. (1953) reported that in rats the degree of tolerance to morphine was partially inhibited when morphine was administered together with nalorphine in contrast to that which is observed when morphine is given alone. Experiments in man indicated that when levallorphan was coadministered with morphine, the development of tolerance to morphine was partially blocked (Eddy et al., 1960). Similar results were reported with levallorphan and
Naltrexone
Naioxone
Receptors
nalorphine in monkeys by Seevers and Deneau (1963) who suggested that since nalorphine was administered prior to morphine, the latter could n o t occupy the receptors responsible for its depressant actions and thus the mechamsms involved in the initiation of physical dependence could not be initiated. More recent studies (Mushlin et al., 1975) showed that concomitant administration of narcotic antagonists inhibited the development of tolerance~ and dependence in chronically
morphinized rats. It has also been shown that naloxone~ antagonizes the dependence which develops a~ter a single injection of morphine in mice (Smits, 1976; Yano and Takemori, 1977; Tremblay et ah, 1976) and in rats (Mushlin and Cochin, 1976; Tremblay et al.,
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1976). Findings contrary to the above have been reported in the literature. For example, Eidelberg and Erspamer (1974) showed that naloxone failed to prevent the development of tolerance and physical dependence in mice induced by a single injection of morphine. Kornetsky and Softer (1974), suggested, on the basis of their work using naloxone to block the effects of morphine in rats, that narcotic receptor occupation by morphine may not be critical for the development of tolerance. Shen and Way (1975) reported that brain concentration of morphine decrease in morphine~tependent mice when they are challenged with naloxone and the changes in brain morphine concentrations following naloxone paralleled the appearance of morphine withdrawal symptoms. It was suggested that the antagonist precipitated withdrawal in morphine dependent rodents may be the result of displacement of morphine by the antagonist from the receptor site in the brain (Shen and Way, 1975). However, Dum et al. (1977) failed to confirm the findings of Shen and Way (1975). It is possible that the antagonist may displace morphine from the central narcotic receptors thereby decreasing the concentration of morphine at the receptor site necessary to produce a certain degree of tolerance and physical dependence upon morphine. This could perhaps explain the lower degree of tolerance or dependence on morphine when morphine is given in combination with a narcotic antagonist compared with the tolerance and dependence produced in absense of an antagonist (Yano and Takemori, 1977). Because of recent interest in naltrexone use in rehabilitating narcotic addicts (Resnick et al., 1977), the present experiments were designed to (a) study the effects of various schedules of naltrexone administration on the development of physical dependence on morphine, and (b) determine if the effects of naltrexone on physical dependence upon morphine are related to the alterations in brain morphine concentration.
H.N. B H A R G A V A
2. Materials and methods
2.1. Assessment of morphine physical dependence Male Swiss Webster mice (Scientific Small Animals Inc., Arlington Heights, Ill.) were housed in laboratories for at least 5 days prior to being used in these studies. The mice weighed 25--30 g at the beginning of each experiment and were allowed access to water and Purina Laboratory Chow ad libitum. Mice were housed eight to a cage and kept at an environmental temperature of 23 + I°C, humidity of ~5 -+ 2% with a 6.00--18.00 h light cycle. Mice were rendered dependent on morphine by the subcutaneous implantation of a specially formulated pellet containing 75 mg of morphine base for three days (Way et al., 1969). Control animals received a placebo pellet for 3 days. All pellet implantation and removal were done between 8.00--9.00 h. Both abrupt withdrawal and naloxone-precipitated withdrawal were used to assess the degree of physical dependence on morphine (Bhargava, 1977b). To precipitate morphine withdrawal, naloxone was injected s.c., 6 h after morphine pellet removal. All the naloxone EDs0's were also determined at the same time to minimize the diurnal variations. Stereotyped jumping has been shown to be a highly characteristic sign of the withdrawal syndrome in rodents (Bhargava, 1977b; Way et al., 1969) and it was used as a quantal response in determining the median effective dose of naloxone (EDs0). Mice were placed on a circular platform immediately after naloxone administration and the percent of mice jumping off the platform within a 15min observation period was noted, using eight to ten mice for each dose of naloxone. The dose response curves were drawn by linear regression analyses. Naloxone EDs0's, potency ratios and their 95% confidence limits were determined by the method of Litchfield and Wilcoxon (1949).
NALTREXONE
A N D PHYSICAL D E P E N D E N C E O N M O R P H I N E
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Physical dependence upon morphine was also assessed by measurement of the responses observed during abrupt morphine withdrawal. Changes in body weight during withdrawal has been used as an index of morphine dependence (Hosoya, 1959; Akera and Brody, 1968; Bhargava, 1977b; Bhargava and Matwysbyn, 1977). After removal of the morphine or placebo pellets, the body weight of each animal was determined over a 36 h period at 3 to 1 2 h intervals. The body weights were expressed as mean -+ S.E.M. using ten mice in each group. Body temperature changes during abrupt withdrawal of morphine has also been used to estimate the intensity of withdrawal or the degree of dependence (Bhargava, 1977b; Bhargava and Matwyshyn, 1977). The rectal temperature of each mouse from morphine or placebo implanted group, receiving saline or naltrexone, was determined with a digital telethermometer (Cole Palmer No. 8502-20, Centigrade thermometer) at various times after the pellet removal. The body temperature was expressed as mean _+ S.E.M. using ten mice in each group.
the jumping response were determined in all the groups. For studies involving body weight and body temperature changes during abrupt withdrawal of morphine, only the schedule 1 treatment described above was used. Mice were injected with saline or naltrexone (20 mg/kg; i.p.). 2 h later one half of the mice in each group were implanted with placebo pellets while the other half were implanted with morphine pellets. The injections of saline or naltrexone were repeated in the animals at 24 and 48 h after the first injection in their respective groups. All the pellets were removed 72 h after their implantation. In studies where naltrexone was injected after the morphine pellet implantation, two schedules were used. In schedule 1, naltrexone (20 mg/kg, i.p.) was administered 24 h after the pellet implantation, while in schedule 2, naltrexone (20 mg/kg) was injected at 24 and 48 h after the pellet implantation. The control group animals received saline. The pellets were. removed as described above and the naloxone EDs0's were determined for all three groups of mice.
2.2. Naltrexone-treatment schedules
2.3. Effect of naltrexone injection on the behavioral change in morphine and placebo implanted mice
The effects of naltrexone administered both before and after morphine pellet implantation were determined. In studies where naltrexone was administered prior to morphine pellet implantation, three schedules were used. In all these schedules, naltrexone (20 mg/kg, i.p.) was injected 2 h prior to morphine pellet implantation. In schedule 1, the same dose of naltrexone was repeated at 24 and 48 h after the pellet implantation. In schedule 2, the same dose was repeated at 36 h after the pellet implantation, whereas, schedule 3 utilized only a single dose of naltrexone. The control groups of mice received saline only. Similar treatment was carried out with placebo pellet implanted mice. Seventy two hours after the implantation, the pellets were removed and 6 h later, naloxone EDs0's for
12 mice were implanted with placebo pellets and another 12 with morphine pellets. 24 h after the implantation 4 morphine dependent mice received saline (0.01 ml/g of body weight) and another 4 received naltrexone (20 mg/kg) i.p. They were immediately put on a circular platform and observed for a period of 15 min. The same animals were injected with saline or naltrexone (20 mg/kg, i.p.) 24 h after the first injection and observed for a 15-rain period as before. Additionally 6 h after pellet removal from 72 h morphine pellet implanted mice were injected with naltrexone (1 mg/kg, i.p.) and observed for 15 min. Similar experiments were carried out with placebo pellet implanted mice.
196
H.N. BHARGAVA
2.4. Determination of brain concentration of morphine In an effort to correlate the observed naltrexone effects with brain morphine concentration, the latter was determined in separate groups of mice treated with various schedules described above. At the end of 72 h after pellet implantation, the mice were sacrificed, their brains removed' and stored frozen at --20°C until analyzed for morphine by the fluorometric procedure of Kupferberg et al. (1964). The glassware was siliconized as previously described (Bhargava, 1977a).
3. Results
3.1. Effect of naltrexone administered prior to morphine pellet implantation on the development of morphine dependence The administration of naltrexone before the morphine pellet implantation inhibited the development of physical dependence on
morphine (table 1). A single injection of naltrexone (20 mg/kg) administered 2 h prior to morphine pellet implantation produced a 14fold increase over the saline controls in the naloxone EDs0 for the jumping response in morphinedtependent mice. Thus the withdrawal jumping response was significantly (P < 0.05) inhibited. When an additional injection of naltrexone (20 mg/kg) was made 24 h after the first injection the increase in naloxone EDs0 was 46 times greater than in the saline control morphine-dependent mice. Similarly, injection of three doses of naltrexone (20 mg/kg), 24 h apart produced a 72-fold increase in the naloxone EDs0. The dose of naloxone as high as 100 mg/kg could not induce jumping behavior in mice implanted with placebo pellets and injected with naltrexone. Administration of naltrexone prevented the weight loss that occurs during chronic morphine treatment. As shown in fig. 1, the weight of animals before placebo or morphine pellet implantation did not differ significantly. Three days after placebo pellet implantation the ani-
TABLE 1 Effect of naltrexone administered prior to morphine pellet implantation on development of morphine dependence.
Treatment I
Naloxone EDs0 pg/kg
Potency ratio
(A) Saline Naltrexone (X3)
15.75 (8.20--30.24) 1131.37 (589.26--2172.23)
71.83 (28.73--179.58) 2
15.75 (10.16--24.41) 727.26 (360.03--1469.07)
46.18 (18.11--117.76) 2
15.75 224.08
14.23
m
(B) Saline Naltrexone (X2)
m
(c) Saline Naltrexone (X1)
(10.16--24.41) (84.24--596.05)
(4.9---41.27) 2
I Mice were injected with saline or naltrexone (20 mg/kg) 2 h prior to morphine pellet implantation (C), the
injections were repeated 36 h later (B), the injections were repeated at 24 and 48 h after the first injection (A). The pellets were removed 72 h after the implantation and naloxone EDs0's were determined 6 h later. Naloxone EDs0 was determined by using 8--10 mice for each of the three doses of naloxone. The values in parentheses represent the 95% confidence limits. The jumping syndrome could not be observed in mice inplanted with placebo pellets with 100 mg/kg dose of naloxone. 2 p < 0.05 vs. the corresponding saline controls.
NALTREXONE
AND
PHYSICAL
DEPENDENCE
ON MORPHINE
197
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Fig. 1. Effect of naltrexone on the body weight changes in morphine dependent mice. Mice were injected i.p. with saline or naltrexone (20 mg/kg) 2 h prior to morphine or placebo pellet implantation. The injections were repeated in their respective g~oups at 24 and 48 h after the first injection. All the pellets were removed 72 h after the implantation and the weight of each animal was recorded for up to 36 h. Each point represents the mean and S.E. (n = I0).
mals gained weight. Naltrexone treatment did not affect the body weights in placebo pelletimplanted animals. Morphine pellet implantation produced a diminished rate of growth compared with naltrexone treated morphine
pellet implanted animals. For the entire 36 h period, of observation, there was no significant difference in the weights of saline or naltrexone treated placebo pellet implanted mice. Morphine pellet-implanted mice injected
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9 12 24 36 Time after pellet removal (hr) Fig. 2. Effect o f naltrexone on the body temperature during abrupt withdrawal in morphine-dependent mice. Mice were injected i.p. with saline or naltrexone (20 mg/kg) 2 h prior to morphine or placebo pellet implantation. The injections were repeated in their respective groups at 24 and 48 h after the f'n~t injection. All the pallets were removed 72 h after the implantation and the body temperature o f each animal was recorded for up to 36 h at various intervals o f time. Each point represents the mean body temperature and S.E. (n = 10).
198
H.N. B H A R G A V A
with saline consistently weighed significantly lower than the naltrexone injected morphinedependent mice and saline or naltrexone injected placebo groups. The naltrexonemorphine group mice also weighed less than the saline-placebo and naltrexone-placebo group, from 3 to 24 h after pellet removal. Naltrexone administration also prevented the development of morphine abstinence hypothermia. In placebo pellet implanted mice naltrexone administration did not affect the body temperature. As can be seen from fig. 2, animals injected with saline and implanted with morphine pellets showed a hypothermic response. A 6% drop in body temperature was noted between 6 and 12 h after pellet removal. Naltrexone treatment prior to and during the pellet implantation resulted in significant reduction in the hypothermic response observed during morphine withdrawal. The body temperatures were significantly ( P < 0.01) lower at 6, 9 and 12 h after the pellet removal compared to placebo groups injected with saline or naltrexone.
3.2. Effect o f naltrexone administered at various times after morphine pellet implantation When naltrexone was administered after the morphine pellet implantation the development of dependence was partially inhibited.
As shown in table 2, naltrexone (20 mg/kg) injected 24 h after the morphine pellet implantation increased the naloxone EDs0 for the jumping response. The naloxone EDs0 in the control group was 22.2 pg/kg in comparison to naltrexone group which had an EDs0 of 796 #g/kg. Thus a 36-fold increase in EDs0 value was noted. Similarly, when the same dose (20 mg/kg) of naltrexone was administered at 24 and 48 h after pellet implantation, a 72-fold increase in the naloxone EDs0 was observed.
3.3. Behavioral observations in morphine-pelleted mice injected with naltrexone Administration of naltrexone to placebo pellet-implanted mice did not produce any observable behavioral change at any time the observations were made. When naltrexone (20 mg/kg) was injected to mice which had morphine pellet for 24 h, a mild abstinence was observed which consisted of a total of 6 jumps for 4 mice within 15-min observation period compared to no jumps in saline treated morphine dependent mice. Naltrexone-treated animals also exhibited more urination and half the animals also showed slight diarrhea. When naltrexone (20 mg/kg) was administered 48 h after the pellet implantation, the total number of jumps for 4 mice recorded in 15 min was 9 and similar effects on urination and
TABLE 2 Effect of naltrexone administered at various times after morphine pellet implantation on the development of morphine dependence. Treatment i
Saline Naltrexone ( X l ) Naltrexone (X2)
Naloxone EDs0 pg/kg 22.2 (13.90--35.52) 795.7 (425.5--1488.0) 1587.4 (1158.7--2174.7)
Potency ratio
35.8 (16.3--78.8) 2 71.5 (40.9--125.1) 2
1 Mice were injected with saline or naltrexone (20 mg/kg, i.p.) 24 h after the morphine pellet implantation (X1). In the second group (X2) the mice received an additional injection of naltrexone at 48 h after the pellet implantation. Naloxone EDs0 was determined by using 8--10 mice for each of the three doses of naloxone. The values in parentheses represent the 95% confidence limits. The jumping syndrome could not be observed in mice implanted with placebo pellets with 100 mg/kg dose of naloxone. 2 p < 0.05 vs. the saline controls.
NALTREXONE
AND PHYSICAL DEPENDENCE
ON MORPHINE
defecation as above were noted. However, when naltrexone injection was repeated at 48 h after implantation in morphine dependent mice that were given an injection of naltrexone at 24 h did not exhibit any sign of morphine withdrawal (viz., jumping, urination, diarrhea). The intensity of the abstinence syndrome induced by naltrexone at 24 or 48 h after pellet implantation was much less compared with that observed when the pellets were removed after 3 days and mice were challenged with a 1 mg/kg dose of naltrexone. In the latter case a t o t a l number Of 26 jumps was recorded in a group of 4 mice within a 15-min observation period. The same mice also showed profuse urination, and excessive defecation and diarrhea.
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3.4. Effect o f naltrexone treatment on brain morphine concentration in morphine-dependent mice Single or multiple naltrexone administration did not change brain morphine concentration in morphine pelleted mice. Similarly, naltrexone administration prior to or after the morphine pellets had been implanted, did not affect the brain morphine concentration. As shown in table 3, the brain morphine concentration in morphine pelleted mice treated with different schedules of naltrexone remained unchanged at approximately 220 ng/g.
4. Discussion
TABLE 3 Effect of naltrexone on brain concentration of morphine-pelleted mice. Treatment
Brain morphine concentration ng/g wet weight (mean +_ S.E.) (n = 4)
Pretreatment 1 Saline Naltrexone ( X l ) Naltrexone (X2) Naltrexone (X3)
221.0 213.0 221.0 220.0
Post-treatment 2 Saline Naltrexone ( X l ) Naltrexone (X2)
229.0 + 22.7 216.0 _+ 28.7 233.0 _+ 21.9
-+ 19.2 _+ 24.4 +- 14.2 + 18.2
1 Pretreatment. Mice were injected with saline or naltrexone (20 mg/kg, i.p.) 2 h prior to morphine pellet implantation ( Xl ), the injections were repeated 36 h later (X2), the injections were repeated at 24 and 4 8 h after the first injection (X3). Mice were sacrificed 72 h after morphine pellet implantation for determination of brain morphine concentration. 2 Post-treatment. Mice were injected with saline or naltrexone (20 mg/kg, i.p.) 24 h after the morphine pellet implantation ( Xl ) . In the second group (X2), mice received an additional injection of naltrexone at 48 h after the pellet implantation. Mice were sacririced as described above for determination of brain morphine concentration.
The present studies clearly demonstrate that naltrexone administration partially inhibits the development of morphine dependence in mice. The inhibition of physical dependence upon morphine was evidenced by increases in the dose of naloxone required to precipitate the withdrawal jumping response in animals. The stereotyped jumping response has been shown to be a highly characteristic sign of morphine withdrawal syndrome and an inverse relationship has been demonstrated for the naloxone EDs0 for the jumping response and the degree of dependence on morphine in the rodents (Way et al., 1969; Bhargava, 1977b). Thus depending upon the schedule of naltrexone administration employed, increases from 14 to 72 fold over the saline control groups in naloxone EDs0's for the jumping response were observed. Although these increases were high, they represented only partial blockades of physical dependence development upon morphine. These studies were substantiated by the studies on abrupt morphine withdrawal. The body weight changes and hypothermia which occur on abrupt morphine withdrawal in morphinedependent rodents (Way et al., 1969; Bhargava, 1977b; Bhargava and Matwyshyn, 1977), were inhibited to a great degree but the corn-
200
plete inhibition was not observed. Thus although the body weight and body temperature in morphine~lependent mice treated with naltrexone were significantly higher thar: those treated with saline, the naltrexonetreated morphine-dependent mice showed slightly lower body weight and temperature than the placebo pellet-implanted mice given either saline or naltrexone. Of major interest was the fact that when naltrexone was injected 24 or 48 h after the induction of physical dependence on morphine, the abstinence syndrome was very mild compared to that observed after antagonistinduced withdrawal in maximally dependent animals (3 days of pellet implantation). This was evidenced by low incidences of withdrawal jumping, defecation, diarrhea and urination. Furthermore, when the injection of naltrexone was repeated 24 h after the first injection, signs of the abstinence syndrome were absent, a fact of much greater importance in a clinical situation. The present studies also indicate that the morphine dependence development can be inhibited not only when the antagonist is given prior to induction of dependence but also when given at any phase of dependence development. The degree of inhibition was similar when naltrexone was administered three times (before and during the morphine pellet implantation) or twice (on days 2 and 3 of pellet implantation) as judged by the increases in the naloxone EDs0's. Recently, plasma pharmakokinetic studies in dogs indicated that the biological half-life of naltrexone is rather short (MacGregor et al., 1977) although it is slightly longer than that of naloxone (Dr. H. Blumberg, personal communication). Naltrexone was therefore, unable to block the narcotic receptor for a long period of time. The present results suggest that occupation of the narcotic receptors by the agonist is important for the genesis of narcotic dependence and support the studies of Yano and Takemori (1977) and Tremblay et al. (1976) who suggested that even the slightest exposure of the receptors to narcotic drugs results in
H.N. BHARGAVA
the development of tolerance and physical dependence. Our results also indicate that the inhibition of morphine dependence development by naltrexone was not associated with alterations in the brain morphine concentration. Shen and Way (1975) reported that naloxone administration to morphine dependent mice from which the pellets had been removed for 6 h produced a precipitous fall in brain morphine concentration. These studies showed that 15 min after administration of naloxone to dependent mice the brain morphine concentration had dropped ten-fold. Furthermore, the brain concentration of morphine was higher at 60 min than at 15 min after naloxone administration. The mechanism of disappearance at 15 min and reappearance at 60 min of displaced morphine is, however, not known. These authors suggested that morphine withdrawal was the result of displacement of morphine from its receptor sites or change in the affinity of the receptors to morphine or naloxone. If such a mechanism is operating then decreased amount of morphine at the receptor site after the antagonist administration would lead to diminished or lower dependence development. In the present study, the brain concentration of morphine in either pretreatment or post treatment of naltrexone were unaffected. Similarly Dum et al. (1977) were unable to observe any fall in brain morphine concentration, as measured by electron capture gas chromatography and radioimmunoassay, in morphine~lependent mice given a challenge dose of naloxone. Attempts have been made to relate opiate tolerance and physical dependence with changes at the receptor level (Collier, 1966). Based on the calculations of the "apparent affinity constants" Tulunay and Takemori (1974a, b) concluded that acute and chronic opiate administration increases the sensitivity for opiate antagonists, suggesting a structural change in the receptors. Hollt et al. (1975) investigated in vivo and in vitro techniques to study changes in opiate receptors during the development of tolerance and physical depen-
NALTREXONE AND PHYSICAL DEPENDENCE ON MORPHINE d e n c e o n m o r p h i n e . N o significant c h a n g e in t h e in vivo " a p p a r e n t a f f i n i t y c o n s t a n t " o f n a l o x o n e in t o l e r a n t rats c o m p a r e d w i t h t h e naive rats c o u l d b e f o u n d . Similar results w e r e obtained on comparison of receptor binding of 3H-etorphine and 3H-naloxone to rat brain h o m o g e n a t e . T h e s e studies indicate t h a t t h e q u a l i t y or t h e q u a n t i t y o f o p i a t e r e c e p t o r binding during the development of tolerance and physical dependence on opiates remains u n a f f e c t e d (Hollt et al., 1 9 7 5 ) . In s u m m a r y , t h e p r e s e n t studies clearly i n d i c a t e t h a t n a r c o t i c a n t a g o n i s t s inhibit t h e d e v e l o p m e n t o f p h y s i c a l d e p e n d e n c e o n narcotics w h e n t h e y are given p r i o r t o or at a n y stage o f d e p e n d e n c e d e v e l o p m e n t . T h e s e e f f e c t s axe p r o d u c e d w i t h o u t altering b r a i n m o r p h i n e c o n c e n t r a t i o n . It is possible t h a t the antagonists induce some conformational changes ( f o r t h e d u r a t i o n o f t h e i r a c t i o n w h i c h is m u c h s h o r t e r t h a n t h e d u r a t i o n o f an agonist like m o r p h i n e ) in t h e r e c e p t o r s o p p o site t o t h a t i n d u c e d b y n a r c o t i c agonists in t h e genesis o f process(es) leading t o t h e development of dependence on morphine.
Acknowledgements The studies were supported by the general funds to the Department of Pharmacognosy and Pharmacology. The author thanks Dr. N.R. Farnsworth for his interest, Mr. George Matwyshyn for his devoted technical assistance and Ms. Juanita Ortiz for her help in preparation of this manuscript. References
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Collier, H.O.J., 1966, Tolerance, physical dependence and receptors, A theory of the genesis of tolerance and physical dependence through drug-induced changes in the number of receptors, Advan. Drug. Res. 3, 171. Dum, J., G. Meyer, V. Hollt, A. Herz and D.H. Catlin, 1977, Inability of naloxone to change brain morphine levels in tolerant mice, European J. Pharmacol. 46,165. Eddy, N.B., M. Piller, L.A. Pirk, O. Shrappe, and S. Wender, 1960, The effect of the addition of a narcotic antagonist on the rate of development of tolerance, Bull. Narc. 12, 1. Eidelberg, E. and R. Erspamer, 1974, Failure of naloxone to prevent acute morphine tolerance and dependence, Arch. Intern. Pharmacodyn. 211, 58. Hollt, V., J. Dum, J. Blassig, P. Schubert and A. Herz, 1975, Comparison of in vivo and in vitro parameters of opiate receptor binding in naive and tolerant dependent rodents, Life Sci. 16, 1823. Hosoya, E., 1959, Some withdrawal symptoms of rats to morphine, Pharmacologist 1, 77. Kornetsky, C. and P. Softer, 1974, Tolerance development in naloxone-morphine treated rats, Federation Proc. 33,501. Kupferberg, H., A. Burkhalter and E.L. Way, 1964, A sensitive fluorometric assay for morphine in plasma and brain, J. Pharmacol. Exptl. Therap. 145, 247. Litchfi'eld, J.T. and F. Wilcoxon, 1949, A simplified method for evaluating dose-effect experiments, J. Pharmacol. Exptl. Therap. 96, 99. MacGregor, T.R., A.E. Staubus, B.E. Morrison, and R. Reunig, 1977, Plasma kinetics of naltrexone and its glucuronide in the dog, Pharmacologist 19, 157. Mushlin, B.E. and J. Cochin, 1976, Tolerance to morphine in the rat: its prevention by naloxone, Life Sci. 18, 797. Mushlin, B.E., P. Griffel, J. Cochin, 1975, Blockade, of the development of physical dependence to morphine by concurrent naloxone administration, Federation Proc. 34,735. Orahovats, P.D., C.A. Winter and E.G. Lehman, 1953, The effect of N-allylnormorphine upon the development of tolerance to morphine in the albino rat, J. Pharmacol. Exptl. Therap. 109, 413. Resnick, R.B., R.S. Kestenbaum, A. Washton and D. Poole, 1977, Naloxone-precipitated withdrawal: a method for rapid induction on to naltrexone, Clin. Pharmacol. Therap. 21,409. Seevers, M.H. and G.A. Deneau, 1963, Physiological aspects of tolerance and physical dependence, in: Physiological Pharmacology, A Comprehensive Treatise, Vol. 1, ecls. W.S. Root and F.G. Hoffman (Academic Press, New York, N.Y.) p. 565. Shen, J.W. and E.L. Way, 1975, Antagonist displace-
202 ment of brain morphine during precipitated abstinence, Life Sci. 16, 1829. Smits, S.E., 1976, Antagonism by naloxone of morphine-induced single-dose dependence and antinociception in mice, Res. Commun. Chem. Path. Pharmacol. 15,689. Tremblay, E., M.-C. Colombel and J. Jacob, 1976, Precipitation et prevention de l'abstinence chez le rat et la souris en ~tat de d~pendance algue. Comparison de la naloxone, de la naltrexone et de la dipr~norphine, Psychopharmacology 49, 41. Tulunay, F.C. and A.E. Takemori, 1974a, The increased efficacy of narcotic antagonists induced by various narcotic analgesics, J. Pharmacol. Exptl.
H.N. BHARGAVA Therap. 190, 395. Tulunay, F.C. and A.E. Takemori, 1974b, Further studies on the alteration of analgesic receptorantagonist interaction induced by morphine, J. Pharmacol. Exptl. Therap. 190,401. Way, E.L., H.H. Loh and F.H. Shen, 1969, Simultaneous quantitative assessment of morphine tolerance and physical dependence, J. Pharmacol. Exptl. Therap. 167, 1. Yano, I. and A.E. Takemori, 1977, Inhibition by naloxone of tolerance and dependence in mice treated acutely and chronically with morphine, Res. Commun. Chem. Pathol. Pharmacol. 16,721.
193
© Elsevier/North-Holland Biomedical Press
THE EFFECTS OF NALTREXONE ON THE DEVELOPMENT OF PHYSICAL DEPENDENCE ON MORPHINE HEMENDRA N. BHARGAVA
Department of Pharmacognosy and Pharmacology, College of Pharmacy, University of Illinois at the Medical Center, Chicago, Illinois 60612, U.S.A. Received 20 January 1978, revised MS received 13 April 1978, accepted 20 April 1978
H.N. BHARGAVA, The effects of naltrexone on the development of physical dependence on morphine, European J. Pharmacol. 50 (1978) 193--202. A single i.p. injection of naltrexone (20 mg/kg) partially inhibited the development of physical dependence upon morphine in mice rendered dependent on morphine by implantation of a pellet containing 75 mg of morphine free base for three days. This was evidenced by an increase in the dose of naloxone (EDs0) required to precipitate withdrawal jumping response. The increase in naloxone EDs0 was much more pronounced when naitrexone was given prior to and during the course of pellet implantation. Inhibition was also observed when naltrexone was administered one day after the morphine pellet implantation, i.e., after some dependence had already developed. Naltrexone administration prior to and during the development of dependence also inhibited, but only partially, the loss in body weight and hypothermic response observed during abrupt withdrawal of morphine in morphine-dependent mice. The inhibitory effect of naltrexone on morphine dependence development was not associated with changes in brain morphine concentration. Brain morphine concentration Morphine dependence
Body weight Hypothermia
1. Introduction The mechanisms involved in the genesis of tolerance and physical dependence on narcotics are still obscure. Narcotic antagonists have been used as pharmacological tools to understand these phenomena. Orahovats et al. (1953) reported that in rats the degree of tolerance to morphine was partially inhibited when morphine was administered together with nalorphine in contrast to that which is observed when morphine is given alone. Experiments in man indicated that when levallorphan was coadministered with morphine, the development of tolerance to morphine was partially blocked (Eddy et al., 1960). Similar results were reported with levallorphan and
Naltrexone
Naioxone
Receptors
nalorphine in monkeys by Seevers and Deneau (1963) who suggested that since nalorphine was administered prior to morphine, the latter could n o t occupy the receptors responsible for its depressant actions and thus the mechamsms involved in the initiation of physical dependence could not be initiated. More recent studies (Mushlin et al., 1975) showed that concomitant administration of narcotic antagonists inhibited the development of tolerance~ and dependence in chronically
morphinized rats. It has also been shown that naloxone~ antagonizes the dependence which develops a~ter a single injection of morphine in mice (Smits, 1976; Yano and Takemori, 1977; Tremblay et ah, 1976) and in rats (Mushlin and Cochin, 1976; Tremblay et al.,
194
1976). Findings contrary to the above have been reported in the literature. For example, Eidelberg and Erspamer (1974) showed that naloxone failed to prevent the development of tolerance and physical dependence in mice induced by a single injection of morphine. Kornetsky and Softer (1974), suggested, on the basis of their work using naloxone to block the effects of morphine in rats, that narcotic receptor occupation by morphine may not be critical for the development of tolerance. Shen and Way (1975) reported that brain concentration of morphine decrease in morphine~tependent mice when they are challenged with naloxone and the changes in brain morphine concentrations following naloxone paralleled the appearance of morphine withdrawal symptoms. It was suggested that the antagonist precipitated withdrawal in morphine dependent rodents may be the result of displacement of morphine by the antagonist from the receptor site in the brain (Shen and Way, 1975). However, Dum et al. (1977) failed to confirm the findings of Shen and Way (1975). It is possible that the antagonist may displace morphine from the central narcotic receptors thereby decreasing the concentration of morphine at the receptor site necessary to produce a certain degree of tolerance and physical dependence upon morphine. This could perhaps explain the lower degree of tolerance or dependence on morphine when morphine is given in combination with a narcotic antagonist compared with the tolerance and dependence produced in absense of an antagonist (Yano and Takemori, 1977). Because of recent interest in naltrexone use in rehabilitating narcotic addicts (Resnick et al., 1977), the present experiments were designed to (a) study the effects of various schedules of naltrexone administration on the development of physical dependence on morphine, and (b) determine if the effects of naltrexone on physical dependence upon morphine are related to the alterations in brain morphine concentration.
H.N. B H A R G A V A
2. Materials and methods
2.1. Assessment of morphine physical dependence Male Swiss Webster mice (Scientific Small Animals Inc., Arlington Heights, Ill.) were housed in laboratories for at least 5 days prior to being used in these studies. The mice weighed 25--30 g at the beginning of each experiment and were allowed access to water and Purina Laboratory Chow ad libitum. Mice were housed eight to a cage and kept at an environmental temperature of 23 + I°C, humidity of ~5 -+ 2% with a 6.00--18.00 h light cycle. Mice were rendered dependent on morphine by the subcutaneous implantation of a specially formulated pellet containing 75 mg of morphine base for three days (Way et al., 1969). Control animals received a placebo pellet for 3 days. All pellet implantation and removal were done between 8.00--9.00 h. Both abrupt withdrawal and naloxone-precipitated withdrawal were used to assess the degree of physical dependence on morphine (Bhargava, 1977b). To precipitate morphine withdrawal, naloxone was injected s.c., 6 h after morphine pellet removal. All the naloxone EDs0's were also determined at the same time to minimize the diurnal variations. Stereotyped jumping has been shown to be a highly characteristic sign of the withdrawal syndrome in rodents (Bhargava, 1977b; Way et al., 1969) and it was used as a quantal response in determining the median effective dose of naloxone (EDs0). Mice were placed on a circular platform immediately after naloxone administration and the percent of mice jumping off the platform within a 15min observation period was noted, using eight to ten mice for each dose of naloxone. The dose response curves were drawn by linear regression analyses. Naloxone EDs0's, potency ratios and their 95% confidence limits were determined by the method of Litchfield and Wilcoxon (1949).
NALTREXONE
A N D PHYSICAL D E P E N D E N C E O N M O R P H I N E
195
Physical dependence upon morphine was also assessed by measurement of the responses observed during abrupt morphine withdrawal. Changes in body weight during withdrawal has been used as an index of morphine dependence (Hosoya, 1959; Akera and Brody, 1968; Bhargava, 1977b; Bhargava and Matwysbyn, 1977). After removal of the morphine or placebo pellets, the body weight of each animal was determined over a 36 h period at 3 to 1 2 h intervals. The body weights were expressed as mean -+ S.E.M. using ten mice in each group. Body temperature changes during abrupt withdrawal of morphine has also been used to estimate the intensity of withdrawal or the degree of dependence (Bhargava, 1977b; Bhargava and Matwyshyn, 1977). The rectal temperature of each mouse from morphine or placebo implanted group, receiving saline or naltrexone, was determined with a digital telethermometer (Cole Palmer No. 8502-20, Centigrade thermometer) at various times after the pellet removal. The body temperature was expressed as mean _+ S.E.M. using ten mice in each group.
the jumping response were determined in all the groups. For studies involving body weight and body temperature changes during abrupt withdrawal of morphine, only the schedule 1 treatment described above was used. Mice were injected with saline or naltrexone (20 mg/kg; i.p.). 2 h later one half of the mice in each group were implanted with placebo pellets while the other half were implanted with morphine pellets. The injections of saline or naltrexone were repeated in the animals at 24 and 48 h after the first injection in their respective groups. All the pellets were removed 72 h after their implantation. In studies where naltrexone was injected after the morphine pellet implantation, two schedules were used. In schedule 1, naltrexone (20 mg/kg, i.p.) was administered 24 h after the pellet implantation, while in schedule 2, naltrexone (20 mg/kg) was injected at 24 and 48 h after the pellet implantation. The control group animals received saline. The pellets were. removed as described above and the naloxone EDs0's were determined for all three groups of mice.
2.2. Naltrexone-treatment schedules
2.3. Effect of naltrexone injection on the behavioral change in morphine and placebo implanted mice
The effects of naltrexone administered both before and after morphine pellet implantation were determined. In studies where naltrexone was administered prior to morphine pellet implantation, three schedules were used. In all these schedules, naltrexone (20 mg/kg, i.p.) was injected 2 h prior to morphine pellet implantation. In schedule 1, the same dose of naltrexone was repeated at 24 and 48 h after the pellet implantation. In schedule 2, the same dose was repeated at 36 h after the pellet implantation, whereas, schedule 3 utilized only a single dose of naltrexone. The control groups of mice received saline only. Similar treatment was carried out with placebo pellet implanted mice. Seventy two hours after the implantation, the pellets were removed and 6 h later, naloxone EDs0's for
12 mice were implanted with placebo pellets and another 12 with morphine pellets. 24 h after the implantation 4 morphine dependent mice received saline (0.01 ml/g of body weight) and another 4 received naltrexone (20 mg/kg) i.p. They were immediately put on a circular platform and observed for a period of 15 min. The same animals were injected with saline or naltrexone (20 mg/kg, i.p.) 24 h after the first injection and observed for a 15-rain period as before. Additionally 6 h after pellet removal from 72 h morphine pellet implanted mice were injected with naltrexone (1 mg/kg, i.p.) and observed for 15 min. Similar experiments were carried out with placebo pellet implanted mice.
196
H.N. BHARGAVA
2.4. Determination of brain concentration of morphine In an effort to correlate the observed naltrexone effects with brain morphine concentration, the latter was determined in separate groups of mice treated with various schedules described above. At the end of 72 h after pellet implantation, the mice were sacrificed, their brains removed' and stored frozen at --20°C until analyzed for morphine by the fluorometric procedure of Kupferberg et al. (1964). The glassware was siliconized as previously described (Bhargava, 1977a).
3. Results
3.1. Effect of naltrexone administered prior to morphine pellet implantation on the development of morphine dependence The administration of naltrexone before the morphine pellet implantation inhibited the development of physical dependence on
morphine (table 1). A single injection of naltrexone (20 mg/kg) administered 2 h prior to morphine pellet implantation produced a 14fold increase over the saline controls in the naloxone EDs0 for the jumping response in morphinedtependent mice. Thus the withdrawal jumping response was significantly (P < 0.05) inhibited. When an additional injection of naltrexone (20 mg/kg) was made 24 h after the first injection the increase in naloxone EDs0 was 46 times greater than in the saline control morphine-dependent mice. Similarly, injection of three doses of naltrexone (20 mg/kg), 24 h apart produced a 72-fold increase in the naloxone EDs0. The dose of naloxone as high as 100 mg/kg could not induce jumping behavior in mice implanted with placebo pellets and injected with naltrexone. Administration of naltrexone prevented the weight loss that occurs during chronic morphine treatment. As shown in fig. 1, the weight of animals before placebo or morphine pellet implantation did not differ significantly. Three days after placebo pellet implantation the ani-
TABLE 1 Effect of naltrexone administered prior to morphine pellet implantation on development of morphine dependence.
Treatment I
Naloxone EDs0 pg/kg
Potency ratio
(A) Saline Naltrexone (X3)
15.75 (8.20--30.24) 1131.37 (589.26--2172.23)
71.83 (28.73--179.58) 2
15.75 (10.16--24.41) 727.26 (360.03--1469.07)
46.18 (18.11--117.76) 2
15.75 224.08
14.23
m
(B) Saline Naltrexone (X2)
m
(c) Saline Naltrexone (X1)
(10.16--24.41) (84.24--596.05)
(4.9---41.27) 2
I Mice were injected with saline or naltrexone (20 mg/kg) 2 h prior to morphine pellet implantation (C), the
injections were repeated 36 h later (B), the injections were repeated at 24 and 48 h after the first injection (A). The pellets were removed 72 h after the implantation and naloxone EDs0's were determined 6 h later. Naloxone EDs0 was determined by using 8--10 mice for each of the three doses of naloxone. The values in parentheses represent the 95% confidence limits. The jumping syndrome could not be observed in mice inplanted with placebo pellets with 100 mg/kg dose of naloxone. 2 p < 0.05 vs. the corresponding saline controls.
NALTREXONE
AND
PHYSICAL
DEPENDENCE
ON MORPHINE
197
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Fig. 1. Effect of naltrexone on the body weight changes in morphine dependent mice. Mice were injected i.p. with saline or naltrexone (20 mg/kg) 2 h prior to morphine or placebo pellet implantation. The injections were repeated in their respective g~oups at 24 and 48 h after the first injection. All the pellets were removed 72 h after the implantation and the weight of each animal was recorded for up to 36 h. Each point represents the mean and S.E. (n = I0).
mals gained weight. Naltrexone treatment did not affect the body weights in placebo pelletimplanted animals. Morphine pellet implantation produced a diminished rate of growth compared with naltrexone treated morphine
pellet implanted animals. For the entire 36 h period, of observation, there was no significant difference in the weights of saline or naltrexone treated placebo pellet implanted mice. Morphine pellet-implanted mice injected
37
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9 12 24 36 Time after pellet removal (hr) Fig. 2. Effect o f naltrexone on the body temperature during abrupt withdrawal in morphine-dependent mice. Mice were injected i.p. with saline or naltrexone (20 mg/kg) 2 h prior to morphine or placebo pellet implantation. The injections were repeated in their respective groups at 24 and 48 h after the f'n~t injection. All the pallets were removed 72 h after the implantation and the body temperature o f each animal was recorded for up to 36 h at various intervals o f time. Each point represents the mean body temperature and S.E. (n = 10).
198
H.N. B H A R G A V A
with saline consistently weighed significantly lower than the naltrexone injected morphinedependent mice and saline or naltrexone injected placebo groups. The naltrexonemorphine group mice also weighed less than the saline-placebo and naltrexone-placebo group, from 3 to 24 h after pellet removal. Naltrexone administration also prevented the development of morphine abstinence hypothermia. In placebo pellet implanted mice naltrexone administration did not affect the body temperature. As can be seen from fig. 2, animals injected with saline and implanted with morphine pellets showed a hypothermic response. A 6% drop in body temperature was noted between 6 and 12 h after pellet removal. Naltrexone treatment prior to and during the pellet implantation resulted in significant reduction in the hypothermic response observed during morphine withdrawal. The body temperatures were significantly ( P < 0.01) lower at 6, 9 and 12 h after the pellet removal compared to placebo groups injected with saline or naltrexone.
3.2. Effect o f naltrexone administered at various times after morphine pellet implantation When naltrexone was administered after the morphine pellet implantation the development of dependence was partially inhibited.
As shown in table 2, naltrexone (20 mg/kg) injected 24 h after the morphine pellet implantation increased the naloxone EDs0 for the jumping response. The naloxone EDs0 in the control group was 22.2 pg/kg in comparison to naltrexone group which had an EDs0 of 796 #g/kg. Thus a 36-fold increase in EDs0 value was noted. Similarly, when the same dose (20 mg/kg) of naltrexone was administered at 24 and 48 h after pellet implantation, a 72-fold increase in the naloxone EDs0 was observed.
3.3. Behavioral observations in morphine-pelleted mice injected with naltrexone Administration of naltrexone to placebo pellet-implanted mice did not produce any observable behavioral change at any time the observations were made. When naltrexone (20 mg/kg) was injected to mice which had morphine pellet for 24 h, a mild abstinence was observed which consisted of a total of 6 jumps for 4 mice within 15-min observation period compared to no jumps in saline treated morphine dependent mice. Naltrexone-treated animals also exhibited more urination and half the animals also showed slight diarrhea. When naltrexone (20 mg/kg) was administered 48 h after the pellet implantation, the total number of jumps for 4 mice recorded in 15 min was 9 and similar effects on urination and
TABLE 2 Effect of naltrexone administered at various times after morphine pellet implantation on the development of morphine dependence. Treatment i
Saline Naltrexone ( X l ) Naltrexone (X2)
Naloxone EDs0 pg/kg 22.2 (13.90--35.52) 795.7 (425.5--1488.0) 1587.4 (1158.7--2174.7)
Potency ratio
35.8 (16.3--78.8) 2 71.5 (40.9--125.1) 2
1 Mice were injected with saline or naltrexone (20 mg/kg, i.p.) 24 h after the morphine pellet implantation (X1). In the second group (X2) the mice received an additional injection of naltrexone at 48 h after the pellet implantation. Naloxone EDs0 was determined by using 8--10 mice for each of the three doses of naloxone. The values in parentheses represent the 95% confidence limits. The jumping syndrome could not be observed in mice implanted with placebo pellets with 100 mg/kg dose of naloxone. 2 p < 0.05 vs. the saline controls.
NALTREXONE
AND PHYSICAL DEPENDENCE
ON MORPHINE
defecation as above were noted. However, when naltrexone injection was repeated at 48 h after implantation in morphine dependent mice that were given an injection of naltrexone at 24 h did not exhibit any sign of morphine withdrawal (viz., jumping, urination, diarrhea). The intensity of the abstinence syndrome induced by naltrexone at 24 or 48 h after pellet implantation was much less compared with that observed when the pellets were removed after 3 days and mice were challenged with a 1 mg/kg dose of naltrexone. In the latter case a t o t a l number Of 26 jumps was recorded in a group of 4 mice within a 15-min observation period. The same mice also showed profuse urination, and excessive defecation and diarrhea.
199
3.4. Effect o f naltrexone treatment on brain morphine concentration in morphine-dependent mice Single or multiple naltrexone administration did not change brain morphine concentration in morphine pelleted mice. Similarly, naltrexone administration prior to or after the morphine pellets had been implanted, did not affect the brain morphine concentration. As shown in table 3, the brain morphine concentration in morphine pelleted mice treated with different schedules of naltrexone remained unchanged at approximately 220 ng/g.
4. Discussion
TABLE 3 Effect of naltrexone on brain concentration of morphine-pelleted mice. Treatment
Brain morphine concentration ng/g wet weight (mean +_ S.E.) (n = 4)
Pretreatment 1 Saline Naltrexone ( X l ) Naltrexone (X2) Naltrexone (X3)
221.0 213.0 221.0 220.0
Post-treatment 2 Saline Naltrexone ( X l ) Naltrexone (X2)
229.0 + 22.7 216.0 _+ 28.7 233.0 _+ 21.9
-+ 19.2 _+ 24.4 +- 14.2 + 18.2
1 Pretreatment. Mice were injected with saline or naltrexone (20 mg/kg, i.p.) 2 h prior to morphine pellet implantation ( Xl ), the injections were repeated 36 h later (X2), the injections were repeated at 24 and 4 8 h after the first injection (X3). Mice were sacrificed 72 h after morphine pellet implantation for determination of brain morphine concentration. 2 Post-treatment. Mice were injected with saline or naltrexone (20 mg/kg, i.p.) 24 h after the morphine pellet implantation ( Xl ) . In the second group (X2), mice received an additional injection of naltrexone at 48 h after the pellet implantation. Mice were sacririced as described above for determination of brain morphine concentration.
The present studies clearly demonstrate that naltrexone administration partially inhibits the development of morphine dependence in mice. The inhibition of physical dependence upon morphine was evidenced by increases in the dose of naloxone required to precipitate the withdrawal jumping response in animals. The stereotyped jumping response has been shown to be a highly characteristic sign of morphine withdrawal syndrome and an inverse relationship has been demonstrated for the naloxone EDs0 for the jumping response and the degree of dependence on morphine in the rodents (Way et al., 1969; Bhargava, 1977b). Thus depending upon the schedule of naltrexone administration employed, increases from 14 to 72 fold over the saline control groups in naloxone EDs0's for the jumping response were observed. Although these increases were high, they represented only partial blockades of physical dependence development upon morphine. These studies were substantiated by the studies on abrupt morphine withdrawal. The body weight changes and hypothermia which occur on abrupt morphine withdrawal in morphinedependent rodents (Way et al., 1969; Bhargava, 1977b; Bhargava and Matwyshyn, 1977), were inhibited to a great degree but the corn-
200
plete inhibition was not observed. Thus although the body weight and body temperature in morphine~lependent mice treated with naltrexone were significantly higher thar: those treated with saline, the naltrexonetreated morphine-dependent mice showed slightly lower body weight and temperature than the placebo pellet-implanted mice given either saline or naltrexone. Of major interest was the fact that when naltrexone was injected 24 or 48 h after the induction of physical dependence on morphine, the abstinence syndrome was very mild compared to that observed after antagonistinduced withdrawal in maximally dependent animals (3 days of pellet implantation). This was evidenced by low incidences of withdrawal jumping, defecation, diarrhea and urination. Furthermore, when the injection of naltrexone was repeated 24 h after the first injection, signs of the abstinence syndrome were absent, a fact of much greater importance in a clinical situation. The present studies also indicate that the morphine dependence development can be inhibited not only when the antagonist is given prior to induction of dependence but also when given at any phase of dependence development. The degree of inhibition was similar when naltrexone was administered three times (before and during the morphine pellet implantation) or twice (on days 2 and 3 of pellet implantation) as judged by the increases in the naloxone EDs0's. Recently, plasma pharmakokinetic studies in dogs indicated that the biological half-life of naltrexone is rather short (MacGregor et al., 1977) although it is slightly longer than that of naloxone (Dr. H. Blumberg, personal communication). Naltrexone was therefore, unable to block the narcotic receptor for a long period of time. The present results suggest that occupation of the narcotic receptors by the agonist is important for the genesis of narcotic dependence and support the studies of Yano and Takemori (1977) and Tremblay et al. (1976) who suggested that even the slightest exposure of the receptors to narcotic drugs results in
H.N. BHARGAVA
the development of tolerance and physical dependence. Our results also indicate that the inhibition of morphine dependence development by naltrexone was not associated with alterations in the brain morphine concentration. Shen and Way (1975) reported that naloxone administration to morphine dependent mice from which the pellets had been removed for 6 h produced a precipitous fall in brain morphine concentration. These studies showed that 15 min after administration of naloxone to dependent mice the brain morphine concentration had dropped ten-fold. Furthermore, the brain concentration of morphine was higher at 60 min than at 15 min after naloxone administration. The mechanism of disappearance at 15 min and reappearance at 60 min of displaced morphine is, however, not known. These authors suggested that morphine withdrawal was the result of displacement of morphine from its receptor sites or change in the affinity of the receptors to morphine or naloxone. If such a mechanism is operating then decreased amount of morphine at the receptor site after the antagonist administration would lead to diminished or lower dependence development. In the present study, the brain concentration of morphine in either pretreatment or post treatment of naltrexone were unaffected. Similarly Dum et al. (1977) were unable to observe any fall in brain morphine concentration, as measured by electron capture gas chromatography and radioimmunoassay, in morphine~lependent mice given a challenge dose of naloxone. Attempts have been made to relate opiate tolerance and physical dependence with changes at the receptor level (Collier, 1966). Based on the calculations of the "apparent affinity constants" Tulunay and Takemori (1974a, b) concluded that acute and chronic opiate administration increases the sensitivity for opiate antagonists, suggesting a structural change in the receptors. Hollt et al. (1975) investigated in vivo and in vitro techniques to study changes in opiate receptors during the development of tolerance and physical depen-
NALTREXONE AND PHYSICAL DEPENDENCE ON MORPHINE d e n c e o n m o r p h i n e . N o significant c h a n g e in t h e in vivo " a p p a r e n t a f f i n i t y c o n s t a n t " o f n a l o x o n e in t o l e r a n t rats c o m p a r e d w i t h t h e naive rats c o u l d b e f o u n d . Similar results w e r e obtained on comparison of receptor binding of 3H-etorphine and 3H-naloxone to rat brain h o m o g e n a t e . T h e s e studies indicate t h a t t h e q u a l i t y or t h e q u a n t i t y o f o p i a t e r e c e p t o r binding during the development of tolerance and physical dependence on opiates remains u n a f f e c t e d (Hollt et al., 1 9 7 5 ) . In s u m m a r y , t h e p r e s e n t studies clearly i n d i c a t e t h a t n a r c o t i c a n t a g o n i s t s inhibit t h e d e v e l o p m e n t o f p h y s i c a l d e p e n d e n c e o n narcotics w h e n t h e y are given p r i o r t o or at a n y stage o f d e p e n d e n c e d e v e l o p m e n t . T h e s e e f f e c t s axe p r o d u c e d w i t h o u t altering b r a i n m o r p h i n e c o n c e n t r a t i o n . It is possible t h a t the antagonists induce some conformational changes ( f o r t h e d u r a t i o n o f t h e i r a c t i o n w h i c h is m u c h s h o r t e r t h a n t h e d u r a t i o n o f an agonist like m o r p h i n e ) in t h e r e c e p t o r s o p p o site t o t h a t i n d u c e d b y n a r c o t i c agonists in t h e genesis o f process(es) leading t o t h e development of dependence on morphine.
Acknowledgements The studies were supported by the general funds to the Department of Pharmacognosy and Pharmacology. The author thanks Dr. N.R. Farnsworth for his interest, Mr. George Matwyshyn for his devoted technical assistance and Ms. Juanita Ortiz for her help in preparation of this manuscript. References
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